Abstract

Huntington’s disease is a neurodegenerative disorder caused by a polyglutamine repeat in the Huntingtin gene (HTT). Although suppressing the expression of mutant HTT (mHTT) has been explored as a therapeutic strategy to treat Huntington’s disease, considerable efforts have gone into developing allele-specific suppression of mHTT expression, given that loss of Htt in mice can lead to embryonic lethality. It remains unknown whether depletion of HTT in the adult brain, regardless of its allele, could be a safe therapy. Here, we report that permanent suppression of endogenous mHTT expression in the striatum of mHTT-expressing mice (HD140Q-knockin mice) using CRISPR/Cas9-mediated inactivation effectively depleted HTT aggregates and attenuated early neuropathology. The reduction of mHTT expression in striatal neuronal cells in adult HD140Q-knockin mice did not affect viability, but alleviated motor deficits. Our studies suggest that non–allele-specific CRISPR/Cas9-mediated gene editing could be used to efficiently and permanently eliminate polyglutamine expansion–mediated neuronal toxicity in the adult brain.

Figure 1

(A) Schematics of the designed HTT-gRNA (T1 and T3). (B) Immunofluorescence showing the transduction of AAV-HTT-gRNA in the striatum and part of the cortex. Ctx, cortex; Str, striatum; CC, corpus callosum; LV, lateral ventricle. Scale bar: 100 μm. (C) Different brain regions from 9-month-old homozygous HD140Q-KI mice injected with AAV-CMV-Cas9 and AAV-HTT-gRNA (T1 and T3) or control-gRNA were analyzed by Western blotting with 1C2 for mHTT and antibodies against Cas9, GFAP, NeuN, p62, caspase 3, and cleaved caspase 3. Vinculin was used as a loading control. Hip, hippocampus. (D) Low- and high-magnification images show the reduction of nuclear HTT and HTT aggregates in the AAV-HTT-gRNA/AAV-CMV-Cas9–injected area in 9-month-old homozygous HD140Q-KI mice compared with the contralateral striatum injected with AAV-HTT-gRNA only. Arrow indicates a remaining cell with nuclear HTT inclusion. Scale bar: 10 μm. The red dashed outline indicates the injected region where mHTT aggregates are markedly reduced. (E) Double immunostaining confirmed the depletion of mHTT in the area expressing HTT-gRNA in the injected striatum of 9-month-old homozygous HD140Q-KI mice. The striatum of a HD140Q-KI mouse injected with AAV-CMV-Cas9 only was used as a control. Scale bar: 20 μm.